The Golgi complex is the central organelle of the secretory pathway involved in transport, post-translational modification and sorting of molecules designed to post-Golgi locations. The mechanism by which molecules move to, through and from the Golgi is the topic of intensive debate. Answers to three questions are required. Do molecules move through the Golgi within cisternae, according to the cisternal maturation hypothesis, or do they move via small vesicles, according to the vesicular transport hypothesis? Second, does cargo exit the Golgi in both the anterograde and retrograde direction via vesicles, tubules or both? Alternatively, all mechanisms may function together, depending on the amount of carbo trafficking and the cell type, cisternal maturation may predominate over vesicular traffic. Finally, we plan to identify the exit sites from the trans-Golgi for the constitutive, regulated and endosomal/lysosomal pathways. Our hypothesis, based on our earlier work, is that exit to each of these pathways is from different Golgi cisternae. These are the questions and hypotheses we will address in the proposed research. Our approach is to first solve the basic 3-D structure the Golgi ribbon at high resolution (5-7nm) using cells and tissues that are optimally preserved by rapid freezing technologies. All three questions will be addressed in each of our three specific aims. S.A. 1 will use NRK cells, which have only a constitutive secretory pathway, and will focus on modulation of the level of traffic through the Golgi. The b- cell of the pancreas will be used in S.A. 2, to focus on the regulated secretory pathway. Finally, the mammary epithelial cell from non- lactating and lactating rodents will be used in S.A. 3 to focus specifically on a cell that is considered a model of cisternal maturation. Statistical evaluation of surface area, volumes, and the number of buds and tubules extending from each cisterna will provide reliable data to evaluate the mechanism of transport through the mammalian Golgi complex.